Project Summary/Abstract Voltage-gated sodium channels (NaVs) are critical transmembrane proteins in skeletal muscle, nerve, heart, and neuronal cells. Malfunction of these proteins has been implicated in numerous health conditions, including epilepsy, arrhythmia, and pain. Saxitoxin (STX) and related guanidinium toxins are highly potent, naturally- occurring compounds which bind select NaV subtypes at low nanomolar potency. One compound, zetekitoxin AB (ZTX), possesses picomolar affinity for several NaVs and is the most structurally complex relative of the guanidinium family of natural products. ZTX is very rare and has only been subject to preliminary study against three of the ten NaVs. The structure of ZTX is also uncertain based on the recorded spectroscopic data, complicating efforts to prepare and study ZTX via chemical synthesis. This research project aims to study the biology and structure of ZTX using a combination of model toxins containing fragments of the putative structure, computational NMR spectroscopy, whole-cell patch-clamp electrophysiology, and molecular biology. Binding affinities for model toxin compounds, using whole-cell voltage clamp techniques against wild-type and mutant NaV isoforms, will provide insight into the impact of key ZTX structural elements on toxin/channel interactions. Computational spectroscopy methods offer the opportunity to rapidly study candidate structures and direct chemical synthesis efforts. This work also aims to achieve the first total synthesis of ZTX in order to unambiguously confirm its assigned structure, with the flexibility to easily produce diverse congeners for biological study. Definitive assignment of the ZTX structure and knowledge of critical toxin/channel binding interactions will lead to small-molecule probes that provide deeper understanding of NaV structure, function, and dynamics. These new small molecules and the study of their interactions with NaVs will lay the groundwork for the future development of compounds capable of selectively modulating dysfunctional NaVs implicated in several serious human channelopathies.